Supplementary Figure 1
Phosphorylation of Myc-Cy(+68) expressed in COS-1 cells. COS-1 cells were transfected with pCMVMyc-Cy(+68) in 6-well plates for 8 hr, washed and labeled with 100mCi 32P-phosphorus (ICN, 400-800mCi/ml) in phosphate-free medium for 16 hr. Cell lysates were prepared (200ml homogenization buffer with protease inhibitors/well), and Myc-Cy(+68) was immunoprecipitated (400ml cell lysate/ 1ml anti-Myc/10ml rProtein A Sepharose (Amersham-Pharmacia)). rProtein A gels were washed 5 times with homogenization buffer, and proteins were eluted with 100ml of 0.17M glycine-HCl, pH 2.3, rapidly neutralized with 1-M tris and analyzed by SDS-PAGE with and without (not shown) DTT. The main Myc-Cy(+68) band is indicated by an arrowhead, and an additional band (38kDa) with 32P-labeling is indicated by an arrow. The heavy and light chains of mouse IgG are indicated by asterisks.
Supplementary Figure 2
SDS-PAGE-immunoblot patterns of altered FLAG-TMCy(+68) expressed in Neuro-2A cells. The previously prepared constructs [17] were used. Cell lysates were prepared and mixted with SDS-PAGE sample mixture with (upper) and without (lower) DTT, boiled and analyzed. Note that TMCy(+68; C3137A) exists in reduced and non-reduced (arrowhead) forms. Blotted with anti-FLAG.
Supplementary Figure 3
Multiple alignment of the Cdh23TMCy regions of vertebrates
Hum 1 MAIIVLAILLFLAAMLFVLMNWYYRTVHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Chi 1 MAIIVLAILLFLAAMLFVLMNWYYRTVHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Mac 1 MAIIVLAILLFLAAMLFVLMNWYYRTVHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Dog 1 MAIIVLAILLFLAAMLFILMNWYYRTVHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Cat 1 MAIIVLAILLFLAAMLFILMNWYYRTVHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Cow 1 MAIIVLAILLFLAAMLFILMNWYYRTVHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Hor 1 MAIIVLAILLFLAAMLFILMNWYYRTIHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Mou 1 MAIIVLAILLFLAAMLFVLMNWYYRTIHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Rat 1 MAIIVLAILLFLAAMLFVLMNWYYRTIHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Arm 1 MAIIVLALLLFLAAMLFILMNWYYRTIHKRKLKAIVAGSA-GNRGFIDIMDMPNTNKYSF 59
Opo 1 MAIIILAILLFLAAMLFILMNWYYRTVHKRKLKAIVAGST-GNRGFIDIMDMPNTNKYSF 59
Xen 1 MAIIILAILLFLAAMLFIFMNWYYRTVHKRKLKAIVAGSLVRNRGFMDILDMPNTNKYSF 60
Zeb 1 MAIIILAVLLFLAAMLFILMNWYYRTVHKRKLKAVVAGST-GNQGLMDILDMPNTNKYTF 59
****.**.*********..*******.*******.****. .*.*..**.********.*
Hum 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Chi 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Mac 60 DGANPVWLDPFCRNLELATQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Dog 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Cat 60 DGANSVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Cow 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEVADLWNSPTRTHGTFGREPAAVKPDDD 119
Hor 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Mou 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPDDD 119
Rat 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWNSPTRTHGTFGREPAAVKPEDD 119
Arm 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEIADLWSSPTRTHGTFGREPAAVKPDDD 119
Opo 60 DGANPVWLDPFCRNLELAAQAEHEDDLPENLSEITDLWNSPARTHGTFGREPSAVKPEDD 119
Xen 61 EGANPVWLDPFCRNMELAAQAEHEDDLPENLSDITDLWNSPARTHGTFGREPTASKPEDD 120
Zeb 60 EGANPVWLDPFCRNLELAAQAEHEDDLPEDLSDIADLWNSPARTHGTFGREPQATKPEDD 119
.***.*********.***.**********.**...***.**.**********.*.**.**
Hum 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Chi 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Mac 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Dog 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Cat 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLK------LFAQRMVQKASS-C--HS 165
Cow 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASSS---HS 176
Hor 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKAAS-C--HS 176
Mou 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Rat 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Arm 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMVQKASS-C--HS 176
Opo 120 RYLRAAIQEYDNIAKLGQIIREGPIKGSLLKVVLEDYLRLKKLFAQRMMQKATS-C--HS 176
Xen 121 RYLRAAIQEYDNIAKLGQIMREGPIKGSLLKVVLDDYMRLKKLLAQRMVHKATTSTGDQS 180
Zeb 120 RYLRAAIQEYDNIAKLGQIMREGPIKGSLLNVVLDDYLRLKKLFAARLVTKSTSQ-GDRS 178
*******************.**********...... *.*.*...*... . .*
Hum 177 SISELIQTELDEEPGDHSPGQGSLRFRHKPPVELKGPDGIHVVHGSTGTLLATDLNSLPE 236
Chi 177 SISELIQTELDEEPGDHSPGQGSLRFHHKPPVELKGPDGIHVVHDSTGTLLATDLNSLPE 236
Mac 177 SISELIQTELDEEPRDHSPGQGSLRFRHKPPVELKGPDGIHVVHGSTGTLLATDLNSLPE 236
Dog 177 SISELIQTELDEEPGERSPGQGSLRFRHKPPTELKGPDGIHMVHGSTGTLLATDLNSLPE 236
Cat 166 SISELIQTELEEEPGEHSPSQGSLRFRHKPPTELKGPDGIHAVHGSTGTLLATDLNSLPE 225
Cow 177 SISELIQTELEEEPGERSPGQGSLRFCHKPPTELRGPDGIHVMHGSTGTLLATDLNSLPE 236
Hor 177 SISELIPTELEEEPGECSPGQGSLRFCHKPPTELKGPDGIHMVHGSTGTLLATDLNSLPE 236
Mou 177 SISELIHTDLEEEPGDHSPGQGSLRFRHKPPMELKGQDGIHMVHGSTGTLLATDLNSLPE 236
Rat 177 SISELIHTDLEEEPGDHSPGQGSLRFRHKPPTELKGPDGIHIVHGSTGTLLATDLNSLPE 236
Arm 177 SISELIQTELEEEPGERSPGQGSLRFRHKPPLELKGPDVMAAAHGSTGTLLATDLNSLPE 236
Opo 177 SISELIQTELEEEVADRSLGQGSLRFRHKQPIELKGPDGIHVVHGSTGTLLAADLNSLPE 236
Xen 181 SVTELIQNEEDED---QSPRQGSLRFKHKQPVELKGPDGIHVVHGSTGTLLTSDINSLPE 237
Zeb 179 SVTELIQSDLDEDDEERIGGRGTLRFKHKLPIELRGPDGVHAVHGSTGTLLTSDLNSLPE 238
*..***.....*...... *.***.**.* **.*.*... .*.******..*.*****
Hum 237 EDQKGLGRSLETLTAAEATAFERNARTESAKSTPLH--KLRDVIMETPLEITEL* 289
Chi 237 EDQKGLGRSLETLTAAEATAFERNARTESAKSTPLH--KLRDVIMETPLEITEL* 289
Mac 237 DDQKGLGRSLETLTAAEATAFERNARTESAKSTPLH--KLRDVIMESPLEITEL* 289
Dog 237 DDQKGLGRSLETLTTAEASAFERNARTESAKSTPLH--KLRDVILESPLEITEL* 289
Cat 226 DDQKGLGRSLETLTTTEASAFERNARTESAKSTPLH--KLRDVILESPLEITEL* 278
Cow 237 DDQKGLGRSLETLTTAEASAFERNARTESAKSTPLH--KLRDVIMESPLEITEL* 289
Hor 237 DDQKGLGRSLETLTAAEASAFERNARTESAKSTPLH--KLRDVIMESPLEITEL* 289
Mou 237 DDQKGLDRSLETLTASEATAFERNARTESAKSTPLH--KLRDVIMESPLEITEL* 289
Rat 237 DDQKGLDRSLETLTASEATAFERNARTESAKSTPLH--KLRDVIMESPLEITEL* 289
Arm 237 DDQKGLGRSLETLTAAEASAFERNARXXXXXXXXXXXXXXXXXXXXXXXXXXXX 290
Opo 237 DDQKVLGRSLETLTAEAGAYNERNARTESAKSTPMH--KMREVIMESPLEITEL* 289
Xen 238 DDQKGLGRSLETLNADNGAFSDRNARTESAKSTPMH--KLRDTIEEGPLEITEL* 290
Zeb 239 DDQRALARSLEALHADGGLYAERNARTESAKSTPLHRNKGSDTLSESPLEITEL* 293
.**..*.****.*...... ************.* *...... *.*******
Multiple alignment of the Cdh23TMCy(+68) regions of vertebrates, obtained from the NCBI and Ensembl Genome Databases. Hum, human; Chi, chimpanzee; Mac, macaque; Hor, horse; Mou, mouse; Arm, armadillo; Opo, opossum; Xen, Xenopus; Zeb, zebrafish. Met1-Met20 is the TM region, and the exon 68 region corresponds to G146-E180 in the human sequence (35 amino acids). Cys residues are shown in red. In the cat, a deletion of 11 amino acids is recognized in the exon 68 region. In armadillo the structure of COOH-terminal is yet to be identified. Cys71 and Cys174 in the figure correspond to Cys3137 and Cys3240 of mouse/human Cdh23(+68), respectively. In the cow, the position 3240 is Ser, and GSH-interacting Cys3269 occurs in exon 69 region (at the position 203 in the figure; also refer to Supplementary Fig. 4). In the horse, another unique Cys exists at the position 3259 (at the position 193 in the figure). Xenopus and zebrafish Cdh23Cy’s contain just one Cys residue at the position of 3137. Note that the position of Cys3240 is occupied by three or four different amino acids in these lower vertebrates.
Supplementary Figure 4
A. DTT-free SDS-PAGE-immunoblot analysis of Myc-Cy(+68; C3240A, R3269C) protein expressed in Neuro-2A cells. The experiment was carried out to determine whether Cys3269 identified in bovine Cdh23Cy is conformation-inducible. The pCMVMyc-Cy(+68; C3240A, R3269C) construct was prepared from pCMVMyc-Cy(+68; C3240A) by mutagenesis. Blotted with anti-Myc. Unlike Myc-Cy(+68; C3240A) protein, Myc-Cy(+68; C3240A, R3269C) emerges in two different forms, suggesting Cys3269 is structural ambivalence-inducible. Note that the faster-migrating non-reduced band (double arrowheads) became weaker with time (0, 60 and 120 min) following treatment of transfected Neuro-2A cells with 1-mM diethyl maleate (DEM). The non-reduced form of Myc-Cy(+68) is indicated by an arrow. Also refer to Figs. 1A and 2. B. Structural ambivalence of FLAG-humanTMCy(+68) and FLAG-humanTMCy(+68; 9626insC). For the constructs and transfection, refer to [17]. Respective non-reduced forms are indicated by arrows. The conditions of SDS-PAGE were the same as for Fig. 1. Blotted with anti-FLAG. For details, see the text.